Chemical Pesticides: Mode of Action and Toxicology - Chapter 3 pps

chapter threePesticides interfering with processes important to all 3.1 Pesticides that disturb energy production 3.1.1 Anabolic and catabolic processes Green plants are anabolic engin

chapter three

Pesticides interfering with

processes important to all

3.1 Pesticides that disturb energy production

3.1.1 Anabolic and catabolic processes

Green plants are anabolic engines that produce organic materials from bon dioxide, other inorganic substances, water, and light energy Neworganic molecules are made by anabolic processes, whereas organic mole-cules are degraded by catabolic processes Plants are also able to degradecomplicated organic molecules, but the anabolic processes dominate Ani-mals, bacteria, and fungi may be called catabolic engines Their task is toconvert organic materials back to carbon dioxide and water Most of theenergy from the catabolism is released as heat, but much is used to build upnew molecules for growth and reproduction Almost all of the energyrequired for these many thousand chemical reactions is mediated throughadenosine triphosphate (ATP), which is broken down to adenosine diphos-phate (ADP) and inorganic phosphate in the energy-requiring biosynthesis.ADP is again rebuilt to ATP with energy from respiration and glycolysis.The basic catabolic processes that deliver ATP are very similar in all organ-isms and are carried out in small intracellular organelles, the mitochondria

car-We should suppose, therefore, that pesticides disturbing the processes are

not very selective, which is indeed the case We find very toxic and lective substances such as arsenic, fluoroacetate, cyanide, phenols, andorganic tin compounds, but also substances with some selectivity due todifferent uptake and metabolism in various organisms Examples are roten-one, carboxin, diafenthiuron, and dinocap.

nonse-3.1.2 Synthesis of acetyl coenzyme A and the toxic mechanism

of arsenic

Acetyl coenzyme A (Ac-CoA) plays a central role in the production of usefulchemical energy, and about two thirds of all compounds in an organism aresynthesized via Ac-CoA Degradation of sugars leads to pyruvate, whichreacts with thiamine pyrophosphate, and the product reacts further withlipoic acid The acetyl–lipoic acid reacts with coenzyme A to give Ac-CoAand reduced lipoic acid Lipoic acid, in its reduced form, has two closelyarranged SH groups that easily react with arsenite to form a cyclic structurethat is quite stable and leads to the removal of lipoic acid (Figure 3.1) Arsenic

is toxic to most organisms because of this reaction It is not used much as apesticide anymore, but in earlier days, arsenicals, such as lead arsenate, wereimportant insecticides Natural arsenic sometimes contaminates groundwa-ter, which led to a tragedy in Bangladesh Wells were made with financialsupport from the World Health Organization (WHO), but their apparentpure and freshwater was strongly contaminated with the tasteless and invis-ible arsenic and many were poisoned In Europe, arsenic is perhaps bestknown as the preferred poison of Agatha Christie’s murderers, but it is alsovaluable for permanent wood preservation, together with copper and othersalts This use, however, also seems to have been terminated because ofarsenic’s bad reputation as a poison and carcinogen

3.1.3 The citric acid cycle and its inhibitors

3.1.3.1 Fluoroacetate

Fluoroacetate is produced by many plants in Australia and South Africa andhas an important function as a natural pesticide for the plants It is highlytoxic to rodents and other mammals In certain parts of Australia, wheresuch plants are abundant, opossums have become resistant to fluoroaceticacid Good descriptions are presented by several authors in Seawright andEason (1993)

The mode of action of fluoroacetate is well understood: it is converted

to fluoroacetyl-CoA, which is thereafter converted to fluorocitric acid Thisstructure analogue to citric acid inhibits the enzyme that converts citric acid

to cis-aconitic acid, and the energy production in the citric acid stops Citricacid, which accumulates, sequesters calcium α-Ketoglutaric acid and there-fore glutamic acid are depleted These changes are, of course, detrimentalfor the organism The nervous system is sensitive to these changes becauseglutamic acid is an important transmitter substance in the so-called

glutaminergic synapses, and calcium is a very important mediator of theimpulses Furthermore, the halt of aerobic energy production is very harmful.

3.1.3.2 Inhibitors of succinic dehydrogenase

Inhibitors of succinic dehydrogenase constitute an important group of gicides In 1966, carboxin was the first systemic fungicide to be marketed Asystemic pesticide is taken up by the organism it shall protect and may killsucking aphids or the growing fungal hyphae The older fungicides are activeonly as a coating on the surface of the plants and do not fight back growingmycelia inside the plant tissue Carboxin and the other anilides, oroxathiin-fungicides, as they are often called, inhibit the dehydrogenation ofsuccinic acid to fumaric acid — an important step in the tricarboxylic acidcycle The toxicity to animals and plants is low in spite of this very funda-mental mode of action The fungicides in this group are anilides of unsatur-ated or aromatic carboxylic acids The first compound in this group to besynthesized was salicylanilide, which since 1930 had a use as a textile pro-tectant

fun-Figure 3.1 The mode of action of arsenic.

HS CH 2

CH 2

CH HS (CH 2 ) 4 CONH 2

S CH 2

CH 2

CH S (CH 2 ) 4 CONH 2

As HO

lipoamide

Other phenylamides with the same mode of action are fenfuram, flutalonil,furametpyr, mepronil, and oxycarboxin.

3.1.4 The electron transport chain and production of ATP

When compounds are oxidized through the tricarboxylic acid cycle (Figure 3.2)

to carbon dioxide and water, electrons are transferred from the compounds

to oxygen through a well-organized pathway, which ensures that the energy

is not wasted and, more importantly, that electrons are not taken up bycompounds that make them into reactive free radicals The electrons are firsttransferred to nicotineamide-adenine dinucleotide (NAD+) and flavine ade-nine dinucleotide (FAD), and from these co-substrates the electrons arepassed on to ubiquinone and further on to the cytochromes in the electrontransport chain Their ultimate goal is oxygen, which is reduced to water.The energy from this carefully regulated oxidation is used to build up ahydrogen ion gradient across the inner mitochondrian membrane, with thelower pH at the inside This ion gradient drives an ATP factory

3.1.4.1 Rotenone

Rotenone is an important insecticide extracted from various leguminousplants It inhibits the transfer of electrons from nicotineamide-adenine(NADH) to ubiquinone

It is also highly toxic to fish and is often used to eradicate unwantedfish populations, for instance, minnows in lakes before introducing trout, or

to eradicate salmon in rivers in order to get rid of Gyrodactilus salaries, an

obligate fish parasite that is a big threat to the salmon population Thenoninfected salmon coming up from the sea to spawn will not be infected

if the infected fish present in the river have been killed before they arrive

Figure 3.2 A simple outline of the citric acid cyclus and the sites of inhibition by the insecticide/rodenticide fluoroacetic acid, and the fungicide carboxin.

CH2C CoA

O F

COOH CO

CH 2

COOH

COOH CHF CCOOH HO

CH 2

COOH

COOH

CH2C-COOH HO

COOH

CH 2

CH 2

CO COOH

CONH Y

fluorocitric acid

3.1.4.2 Inhibitors of electron transfer from cytochrome b to c 1

The strobilurins are a new class of fungicides based on active fungitoxicsubstances found in the mycelia of basidiomycete fungi The natural prod-ucts, such as strobilurin A and strobilurin B, are too volatile and sensitive

to light to be useful in fields and glasshouses However, by manipulatingthe molecule, notably changing the conjugated double bonds that make themlight sensitive, with more stable aromatic ring systems, a new group offungicides have been developed in the last decade At least four are on themarket (azoxystrobin, famoxadone, kresoxim-methyl, and trifloxystrobin).Their mode of action is the inhibition of electron transfer from cytochrome

b to cytochrome c1 in the mitochondrial membrane They are supposed tobind to the ubiquinone site on cytochrome b

The reactions inhibited by strobilurin fungicides:

The fungicides are very versatile in the contol of fungi that have becomeresistant to the demethylase inhibitor (DMI) fungicides described later Theyhave surprisingly low mammalian toxicity, but as with many other respira-tory poisons, they show some toxicity to fish and other aquatic organisms.They may also be toxic to earthworms In fungi they inhibit spore germina-tion The structures show the natural products strobilurin B and azox-ystrobin, which has been marketed since 1996

CH3

CH 3 O

OCH3O

CH 3 O

3.1.4.3 Inhibitors of cytochrome oxidase

Cyanide may still have some use against bedbugs and other indoor pests inspite of its high toxicity to man, but in the past it was used much more Inthe 19th century, doctors prescribed it as a sedative and, of course, caused

a lot of fatal poisoning (Otto, 1838) The recommended treatment was to letthe patient breathe ammonia Today we have very efficient antidotes, such

as sodium nitrite and amyl nitrite They cause some of the Fe++ of hemoglobin

to be oxidized to Fe+++, which then binds the CN– ion Cyanide inhibits thelast step in the electron transport chain catalyzed by cytochrome oxidase bybinding to essential iron and copper atoms in the enzyme Cyanide is veryfast acting and blocks respiration almost totally

Phosphine is used extensively as a fumigant and is very efficient in thecontrol of insects and rodents in grain, flour, agricultural products, andanimal foods It is used to give continual protection during shipment ofgrain The gas is flammable and very unstable and is changed into phospho-ric acid by oxidation By using pellets of aluminum phosphide at the top ofthe stored product, phosphine is slowly released by reacting with moisture.Other phosphine salts are also used Phosphine is reactive and is probablyinvolved in many reactions, but the inhibition of cytochrome oxidase is themost serious The gas is very toxic to man, but residues in food cause noproblems because it is oxidized rapidly

3.1.4.4 Uncouplers

As discussed in Chapter 2, Section 1.4, uncoupling energy production andrespiration is one of the fundamental toxic mechanisms Weak organic acids

or acid phenols can transport H+ ions across the membrane so that energy

is wasted as heat, and not used to produce ATP

The name uncouplers arose from their ability to separate respiration from

ATP production Even when ATP production is inhibited, the oxidation ofcarbohydrates, etc., can continue if an uncoupler is present Although theuncouplers are biocides, in principle toxic to all life-forms, many valuablepesticides belong to this group However, few of them are selective, and theyhave many target organisms The inner mitochondrial membranes are theirmost important sites of action, but chloroplasts and bacterial membraneswill also be disturbed

Figure 3.3 shows how weak acids can transport H+ ions across the brane

mem-Pesticides with this mode of action include such old products as thedinitrophenols (dinitroorthocreosol [DNOC], dinoterb, and dinoseb) andother phenols such as pentachlorophenol and ioxynil DNOC is a biocideuseful against mites, insects, weeds, and fungi The mammalian toxicity israther high, with a rat oral LD50 (lethal dose in 50% of the population) of

25 to 40 mg/kg of the sodium salt The typical symptom is fever, which is

AIP+3H O2 →AI OH( )3+PH3

in accordance with its biochemical mode of action The uncouplers havebeen tried in slimming treatments with fatal consequences.

Dinocap is an ester that is taken up by fungal spores or mites It ishydrolyzed to the active phenol It has low toxicity to plants and mammals.Dinocap is a mixture of several dinitrophenol esters, and the structure ofone is shown

Ioxynil is a more important uncoupler that is widely used as an cide It acts in both mitochondria and chloroplasts Bromoxynil is similar tothe ioxynil, but has bromine instead of iodine substitutions

herbi-3.1.5 Inhibition of ATP production

ATP is produced from ADP and phosphate by an enzyme, ATP synthase,located in the inner mitochondrian or chloroplast membrane The energy isdelivered from a current of H+ ions into the mitochondrian matrix Someimportant pesticides inhibit this enzyme, leading to a halt in ATP production

Figure 3.3 Transportation of H + ions across a biological membrane by a weak acid.

acid side

NO 2

O 2 N

dinoterb

O CH

CN

OC(CH2)6CH3I I

CN O

ioxynil ioxyniloctanoate

as fungicide, algicide, or molluscicide The toxicity of these compounds tofish is very high, but they have moderate toxicity to rodents The data in

Table 3.1 are taken from The Pesticide Manual (Tomlin, 2000).

Tributyltin and tributyltin oxide are still used on boats and ships toprevent growth of barnacles They are extremely toxic for many invertebrates

in the sea, notably some snails whose sexual organs develop abnormally Inthese snails the female develops a penis In oysters and other bivalves, theirshells become too thick Tributyltin must be regarded as one of the mostserious environmental pollutants, but contrary to the lower analogues, tri-methyltin and triethyltin, they are not very toxic to man and other mammals.Trimethyltin is of considerable interest for neurotoxicologists because it leadsspecifically to atrophy of the center for short-term memory, the hippocam-pus The ethyl analogue has other serious detrimental effects on the brain

Table 3.1 Diafenthiuron and Organotin Compounds Used as Pesticides

Pesticide

Fish (Various Species) LC50 (24–96 h) (mg/l)

Daphnia EC50 (48 h) (mg/l)

Rodents (Various Species or Sex) Oral LD50 (mg/kg)

Source: Data from Tomlin, C., Ed 2000 The Pesticide Manual: A World Compendium British Crop

Protection Council, Farnham, Surrey 1250 pp.

H2O2 may be produced as a by-product in the catalytic cycle of the CYPenzymes described later Diafenthiuron therefore becomes more active insunshine, and piperonyl butoxide that inhibits CYP enzymes makesdiafenthiuron less toxic However, some CYP enzymes are also important inthe detoxication of diafenthiuron, as shown in Figure 3.4 Diafenthiuron may

Figure 3.4 Activation and detoxication of diafenthiuron.

O

N C N S

CH CH

CH CH

CH CH

toxic oxidation product

NH C N OCH3

CH 3

O R

be used against mites, aphids, and other insects on several crops such ascotton, vegetables, and fruit.Table 3.1 shows that it has very high fish toxicity.

3.1.5.3 Summary

The mitochondrial poisons include such a variety of compounds with somany different activities on the organismic level that a summary may help.Figure 3.5 and Table 3.2 may help to identify the site of reaction

In Figure 3.5, the arrows show the electron flow When reaching oxygenthe normal way, water is formed, while in the sideline via paraquat, super-oxide radicals are formed

3.2 Herbicides that inhibit photosynthesis

About half of all herbicides inhibit photosynthesis Most of them disturb oneparticular process, i.e., the transfer of electrons to a low molecular quinonecalled plastoquinone The inhibition occurs by the binding of the inhibitor

to a specific protein called D1 that regulates electron transfer This proteinhas 353 amino acid residues and spans the thylakoid membrane in thechloroplasts In atrazine-resistant mutants of certain plants, a serine residue

at position 264 in the D1 protein of the wild type has been found to besubstituted by glycine It is now possible to replace the serine 264 withglycine by site-directed mutagenesis in its gene and to reintroduce the alteredgene to engineer atrazine resistance in plants

The inhibitors of photosynthesis are all nitrogen-containing substanceswith various structures They may be derivatives of urea, s-triazines, anilides,

Figure 3.5 The site of inhibition of various pesticides in the citric acid cycle and the electron transport chain.

NADH

UQ Cyt b Cyt C 1

Cyt C Cyt ( a + a 3 )

Site of action in the electon transport chain

as-triazinones, uraciles, biscarbamates, pyridazinones, triles, nitrophenols, or benzimidazols We shall describe just a few of them andgive a very brief outline of the photosynthetic process Textbooks of cell biology,biochemistry, and plant physiology (e.g., Alberts et al., 2002; Nelson and Cox,2000; Taitz and Zaiger, 1998) describe the process in detail The action of theherbicides may be read in more detail in Fedke (1982) or Devine et al (1993).

hydroxybenzoeni-In photosynthesis, light energy is trapped and converted to chemicalenergy as reduced coenzymes (e.g., nicotineamide-adenine dinucleotidephosphate [NADPH]), triphosphates (e.g., ATP), and O2 Oxygen is a poi-sonous waste product in plants, although they also need some oxygen inmitochondrial respiration

The chlorophyll takes up light energy (photons) directly or throughso-called antennae molecules (All colored substances take up light energy,but convert it to heat and not to chemical energy.) Electrons that jump toanother orbit requiring more energy take up the energy and are said to havebecome excited Such excited electrons may be lost by being taken up by anacceptor molecule, leaving chlorophyll as a positively charged ion Accord-ing to this scheme, chlorophyll will have three different states: the normalform that can pick up light energy, the excited molecule that is a very strongreducing substance, and the positively charged ion that is a very strong oxidant.The reducing power in the excited chlorophyll molecule is used to produceATP and NADPH, while the oxidation power of the chlorophyll ion is used to

Table 3.2 Site of Action of Some Mitochondrial Poisons

Inhibition of acetyl-CoA

synthesis

Inhibition of akonitase Fluoroacetic acid

(fluorocitrate)

Most animals Inhibition of succinic

dehydrogenase

Salicylanilide and oxathiin fungicides

Fungi Inhibition of NADH

dehydrogenase

Inhibiting cytochrome b Strobinurins Fungi

Inhibiting cytochrome oxidase Cyanide

Phosphine

All aerobic organisms

pH gradient in mitochondrial

membranes (uncouplers)

Inhibitors of ATP synthase in

the mitochondrial membrane

Organonotin compounds Diafenthiuron metabolite

Fungi, mites, aquatic organisms; some have high mammalian neurotoxicity Insects, fish Superoxide generators

Takes electrons from the

transport chain and delivers

them to O2

Copper ions Paraquat

Most organisms Most aerobic organisms

produce ATP and oxygen ATP production is an indirect process coupled to the

pH gradient between the inside and outside of the thylakoid membrane.The photosynthetic apparatus is situated on and in the thylakoid mem-brane

Four different and complicated protein complexes carry out the sary chemical reactions: photosystem II, the cytochrome b6f complex, pho-tosystem I, and ATP synthase These complexes are precisely oriented andfixed in the membrane In addition, there are the plastoquinones, whicheasily undergo a redox cycle and can swim in the membrane’s lipid phase

neces-A manganese-containing complex in photosystem II is involved in the ting of water and the generation of electrons and molecular oxygen A small,copper-containing protein, plastocyanine (PC), is involved in transfer ofelectrons from cytb6f to photosystem I

split-The thylakoid matrix is an extensive internal membrane system insidethe chloroplasts, which are small organelles in the plant cells The innerlumen of the membrane system maintains a pH of 5, while the outer com-partment, called stroma, has a pH of 8 The energy picked up from thephotons is used to establish and maintain this difference

The chlorophyll pigments in photosystem II, organized in a structurecalled P680, catch energy from a photon and become excited The electron

is then transferred to a molecule called pheophytin and then to a tyrosineresidue in protein D1 called the reaction center The oxidized form of plas-toquinone (PQ) has a specific binding site on protein D1, where it is reducedand then diffuses to the lumen side of the membrane (now as plastohydro-quinone (PQH2)) Here it binds to an iron–sulfur protein in the cytochrome

b6f complex and reduces it The hydrogen ions released in this process aredelivered to the inside of the membrane The plastoquinone/plastohydro-quinone is thus functioning as a proton pump driven by light-excited elec-trons A summary of the process is shown in Figure 3.7

Figure 3.6 Blocking the passage of electrons from light-excited chlorophyll to toquinone by herbicides leads to production of singlet oxygen and electrons that may produce free radicals.

plas-Chlorophyll*

Chlorophyll Chlorophyll

to produce plastohydroquinone)

O 2

A simplified scheme of the redox cycle of plastoquinone is shown inFigure 3.8 The quinoid structure and the isoprene side chain make it possiblefor plastoquinone to take up one electron at a time, producing rather stablesemiquinone radicals (which is not shown in the figure), and there areprobably at least two plastoquinones involved.

The reduced cytochrome f delivers the electron to plastocyanine, a per-containing, low-molecular-weight soluble protein, and then to special

cop-Figure 3.7 Schematic representation of photosynthesis and the site of action of D1 blockers and paraquat.

Figure 3.8 Schematic representation of the redox cycle of plastoquinone.

Mn 2H 2 O O2+ 4H+

4

PQ PQH2

2H+

2e Delivered to Cyt f Received from D 1

Lumen side Stroma side

chlorophyll pigments in photosystem I (P700) The P700 can be excited by anew photon and deliver the electron to an iron-containing protein calledferredoxin The reduced ferredoxin delivers the electrons to NADP+ to pro-duce NADPH, or to a minor pathway that reduces nitrate to ammonia atthe outer membrane surface Some important herbicides (paraquat anddiquat) can snatch the electrons before the delivery to ferredoxin and gen-erate free radicals.

The chlorophyll ion in P680+ takes electrons from water, via a nese-containing enzyme complex, and is reduced to the neutral unexcitedstate, ready to pick up new photons Water is then split to oxygen andhydrogen ions Oxygen is a toxic waste product, while the hydrogen ionscontribute to the buildup of the pH difference across the membrane.ATP is produced from ADP and phosphate by ATP synthase, an enzymelocated in the membrane The difference in hydrogen ion concentrationbetween the inside and outside is used as the energy source Because thereare approximately 1000 times more hydrogen ions on the inside than on theoutside of the membrane, the hydrogen ions will tend to diffuse out Thiswould waste energy, so instead, hydrogen ions are forced to flow through aspecial proton channel in the ATP synthase that uses the energy from thehydrogen ion flow to produce ATP ATP synthase is very similar in chloro-plasts and mitochondria

manga-In summary, there are four main types of herbicides that disturb thephotosynthetic apparatus:

1 Weak organic acids that destroy the hydrogen ion concentration dient between the two sides of the membrane

gra-2 Free radical generators

3 Compounds that bind to the D1 protein at (or near) the binding site

plastoquinone-4 Substances that destroy or inhibit synthesis of protecting pigmentssuch as carotenoids

3.2.1 Weak organic acids

Weak organic acids with a pK value between pH 5 and 8, or close to thesevalues, will cause leakage of hydrogen ions if the acid dissolves in thethylakoid membrane Ammonia also has this effect as a result of the reaction

NH4+ →← NH3+ H+ Instead of producing ATP, heat will be generated Theseso-called uncouplers will act similarly in mitochondria, chloroplasts, andbacterial cell membrane They may therefore also be toxic for animals andmicroorganisms, and some of them are described under mitochondrial poisons

3.2.2 Free radical generators

These are a type of herbicide that is able to steal the electron on its longroute from water to NADP+ The most important herbicides in this category